In vitro characterization of [125I]HY-3-24, a selective ligand for the dopamine D3 receptor

Introduction Dopamine D3 receptor (D3R) ligands have been studied for the possible treatment of neurological and neuropsychiatric disorders. However, selective D3R radioligands for in vitro binding studies have been challenging to identify due to the high structural similarity between the D2R and D3R. In a prior study, we reported a new conformationally-flexible benzamide scaffold having a high affinity for D3R and excellent selectivity vs. D2R. In the current study, we characterized the in vitro binding properties of a new radioiodinated ligand, [125I]HY-3-24. Methods In vitro binding studies were conducted in cell lines expressing D3 receptors, rat striatal homogenates, and rat and non-human primate (NHP) brain tissues to measure regional brain distribution of this radioligand. Results HY-3-24 showed high potency at D3R (Ki = 0.67 ± 0.11 nM, IC50 = 1.5 ± 0.58 nM) compared to other D2-like dopamine receptor subtypes (D2R Ki = 86.7 ± 11.9 nM and D4R Ki > 1,000). The Kd (0.34 ± 0.22 nM) and Bmax (38.91 ± 2.39 fmol/mg) values of [125I]HY-3-24 were determined. In vitro binding studies in rat striatal homogenates using selective D2R and D3R antagonists confirmed the D3R selectivity of [125I]HY-3-24. Autoradiography results demonstrated that [125I]HY-3-24 specifically binds to D3Rs in the nucleus accumbens, islands of Calleja, and caudate putamen in rat and NHP brain sections. Conclusion These results suggest that [125I]HY-3-24 appears to be a novel radioligand that exhibits high affinity binding at D3R, with low binding to other D2-like dopamine receptors. It is anticipated that [125I]HY-3-24 can be used as the specific D3R radioligand.


Introduction
The dopamine D3 receptor (D3R) is an important receptor in the brain and is one of the members in D2-like receptor family, which also includes dopamine D2 (D2R) and dopamine D4 receptors (D4R).D3R is a G-protein coupled receptor (GPCRs) and inhibits cAMP signaling through Gα i/o G-proteins (Robinson and Caron, 1996;Missale et al., 1998).D3R is predominantly located in limbic areas such as the ventral pallidum (Diaz et al., 1994(Diaz et al., , 1995)), nucleus accumbens (NAc), olfactory tubercle (OT), and islands of Calleja (ICj), and has a lower density in the dorsal striatum in rat brain (Bouthenet et al., 1991;Levesque et al., 1992).The regional distribution of the D3R led to the conclusion that it may play an important role in neurological and neuropsychiatric disorders such as schizophrenia (Gurevich et al., 1997), Parkinson's disease (Brooks, 2000;Elgueta et al., 2017), and drug addiction (Heidbreder et al., Lee et al. 10.3389/fnins.2024.1380009Frontiers in Neuroscience 02 frontiersin.org2005; Sokoloff et al., 2006).Thus, targeting D3R has been pursued as a potential treatment of neuropsychiatric disorders, but the development of D3R ligands has faced numerous challenges over the past 2 decades.As a member of the D2-like receptor family, the D3R has both structural and pharmacological similarities to the other members, especially the D2R (Missale et al., 1998).The D2R and D3R share 78% amino acid sequence homology in the transmembrane domains (TM) (Giros et al., 1990;Sokoloff et al., 1990) and follow the same signaling pathway (Robinson and Caron, 1996;Cho et al., 2008).Furthermore, there are brain regions that express both D2 and D3 receptors but with different receptor densities (Missale et al., 1998;Sun et al., 2012).
Several radioligands such as [ 3 H]WC-10 (Xu et al., 2009(Xu et al., , 2010)), [ 3 H]spiperone (Vile et al., 1995;Zhen et al., 2010), [ 125 I]IABN (Luedtke et al., 2000), and [ 125 I](R,S)-trans-7-OH-PIPAT (Burris et al., 1994;Stanwood et al., 2000) have been used in radioligand binding assays and autoradiography studies of the D3 receptor.However, all of these radioligands have an affinity for D2 receptors, which makes it difficult to measure the density of D3 receptors in tissues where both D2 and D3 receptors are present.For example, [ 3 H]WC-10, the most D3-selective of the radioligands described above, requires a duo-radioligand study with [ 3 H]raclopride and the use of simultaneous equations to calculate the fractional D2 and D3 occupancy of each radioligand to D2 and D3 receptors quantify D3 receptor density in tissue sections (Xu et al., 2009).Therefore, there is a need to develop a highly D3 selective radioligand that is capable of quantifying D3 receptor density without the need of complex calculations.
Metoclopramide was developed as a dopamine receptor antagonist (Peringer et al., 1975;Elliott et al., 1978;Stanley and Wilk, 1979) and was approved by the Food and Drug Administration (FDA) for use for the treatment of gastroparesis.However, it has limitations for use in other therapeutic applications such as neurological and neuropsychiatric disorders because of its low affinity and selectivity D2 and D3 receptors (K i = 21 ± 1 nM for hD2R and K i = 27 ± 3 nM hD3R; Yang et al., 2000).To address these limitations, we designed a new scaffold based on metoclopramide in which an arylalkyl moiety was introduced to enable an interaction with the secondary binding site of the D3R (Kim et al., 2023).This new scaffold was found to have a high affinity for the D3R and excellent selectivity vs. the D2R.The structure-activity relationship study also identified the bromo analog (compound 6 in Scheme 1) as having a high affinity and selectivity for the D3R (K i = 107 ± 5 nM for hD2R and K i = 1.1 ± 0.1 nM hD3R).In the current study, we describe the properties of a new radioligand, [ 125 I]HY-3-24 that possesses an enhanced affinity for targeting D3R, an increased D3R selectivity vs. D2R, and low off-target binding to other CNS targets.The pharmacological profile of this new radioligand was characterized by in vitro binding studies in tissue homogenates and tissue sections.Our results show that [ 125 I]HY-3-24 is a novel radioligand for use in in vitro binding and autoradiography studies of the D3R.

Radioligand binding assays 2.2.1 D2R/D3R/D4R binding assay
The dopamine receptor binding assay was performed according to previous studies (Luedtke et al., 2000;Vangveravong et al., 2006;Reilly et al., 2017), and human D2R or D3R or D4 transfected HEK293 cells were used for dopamine receptor binding assay.The cells were grown in Dulbecco's Modified Eagle Medium including 10% FBS, 0.1% Penn Strep, 50 mg/mL of geneticin (G418) for human D2R or D3R transfected HEK293 cells, and 10 mg/mL of puromycin for human D4 HEK293 cells at 5% CO 2 , 37°C.The cells were collected by centrifuge (6,000 × g, 10 min), and the supernatant was discarded.Cold homogenization buffer (50 mM Na Hepes, 0.1 mM EDTA, 1 mM DTT, pH 7.4) was added into the pellet and homogenized by a polytron (Brinkmann Instruments, Westbury, NY, United States), and centrifuged at 40,000 × g for 10 min.This process was repeated two more times to get final pellet, and the membranes were then resuspended with HB buffer including 5 mM MgCl 2 and stored at −80°C until use.
HY-3-24 was prepared in 10 −5 to 10 −11 M concentration range with assay buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM EDTA, pH 7.5).Membrane homogenates (5-15 μg) and [ 125 I]IABN (approximately 0.5 nM) was added into serial dilutions of the compound and incubated at 37°C, for 60 min.Total binding volume was 150 μL, and 20 μM of (+)-butaclamol was used to define non-specific binding.Cold buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.5) was added into the reaction tube to terminate binding assay, and mixture was quickly filtered through a Schleicher and Schuell No.32 filter (GE Heathcare Bio-Sciences, Pittsburgh, PA, United States).The membrane was washed with cold buffer (three times), and collected filters were counted on a gamma counter (GMI, Minnesota, United States).The results were analyzed by nonlinear regression, and the data were reported mean ± SEM values by three independent experiments.
For sigma-2 binding assay, HY-3-24 prepared in 10 −5 to 10 −11 M concentration range was mixed with [ 125 I]RHM4 (50 μL,~200,000 cpm) and rat liver homogenates (15 μg/100 μL).The mixture was vortexed briefly and incubated at room temperature (RT) for 90 min.The [ 125 I] RHM4-bound membrane was collected through Whatman CF/C filters by 24-well harvester (Brandel Inc., Maryland, United States), and washed with 4 mL of cold buffer.The filters were counted on a gamma counter (PerkinElmer, Massachusetts, United States).Cold RHM4 was used for defining non-specific binding.The K i value was obtained by PRISM 9.

β-arrestin recruitment assay for D3R
Chinese hamster ovary CHO-K1 cells (CHO-K1) which were over expressed human D3R were cultured in assaycomplete™ cell culture kit 107 (DiscoverX, Fremont, CA, United States).Cells were seeded at a density of 25,000 cells per well of 96-well plate, and incubated at 5% CO 2 , 37°C.After 46 h, HY-3-24 was (10 −5 to 10 −11 ) in phosphatebuffered saline (PBS) was added to the cells.The plates were incubated for 30 min at 5% CO 2 , 37°C for the agonist binding assay; for the antagonist assay, cells were treated with 30 nM (EC 80 ) of dopamine and then the mixture was incubated for 90 min.PathHunter™ β-arrestin recruitment assay kit (DiscoverX, Fremont, CA, United States) was added to each well, and the plate was incubated for 80 min at RT under the dark condition.The chemiluminescent signal was measured on a PerkinElmer Enspire plate reader (PerkinElmer, Boston, MA, United States).Data were analyzed by Prism followed by non-linear regression.

Radiochemistry
Test labeling was performed using two different precursors 7 or 8. First, 100 μL of 100 mM of Cu(pyridine) 4 (OTf) 2 in MeCN solution and 100 μL of 100 mM of 3,4,7,8-tetramethyl-1,10-phenanthroline in MeOH solution were mixed to make 50 mM catalyst mixture.To a solution of 7 or 8 (100 μg, 0.2 μmol) in 50 μL of MeOH, 100 μL of 50 mM catalyst mixture was added followed by 2 μL of Na[ 125 I] (~ 2.22 MBq).The reaction mixture was incubated at room temperature for 30 min or heated at 100°C for 10 min.300 μL of 0.1 M ammonium acetate buffer (pH 4.6) was added for quenching, and each reaction was assessed by HPLC [stationary phase: Luna ® 5 μm C18 100 Å, 10 mm × 250 mm, mobile phase: 68% 0.1 M ammonium acetate buffer (pH 4.6) in MeCN, wavelength 254 nm, flow rate: 4 mL/ min, retention time: 8.6 min].For scale-up radiolabeling studies, 8 was used as the precursor.To a solution of 8 (200 μg, 0.4 μmol) in 50 μL of MeOH, 100 μL of 50 mM catalyst mixture and 100 μL of Na[ 125 I] (318 MBq) in 0.1 N aq NaOH solution were added.The reaction mixture was heated at 100°C for 10 min and incubated at room temperature for 30 min.After the completion of the reaction, the mixture was quenched by 0.6 mL of aqueous mobile phase and the crude product was purified by preparative HPLC (stationary phase: Luna ® 5 μm C18 100 Å, 10 mm × 250 mm, mobile phase: 75% 0.1 M ammonium acetate buffer in MeCN, wavelength 254 nm, flow rate: 4 mL/min, retention time: 22.5 min).The product fraction was collected in a tube containing 0.6 mL of ammonium hydroxide and 30 mL of water.The basic solution was passed on tC18 cartridge, and the cartridge was washed by 5 mL of water.The final product was eluted using 1 mL of ethanol.Radiochemical purity and molar activity was measured by analytical HPLC (stationary phase: Luna ® 5 μm C18 100 Å, 10 mm × 250 mm, mobile phase: 68% 0.1 M ammonium acetate buffer in MeCN, wavelength 254 nm, flow rate: 4 mL/min, retention time: 8.4 min).

Distribution coefficient (LogD 7.4 )
In a separatory funnel, 10 mL of n-octanol and 20 mL of 0.01 M phosphate buffer (pH 7.4) solution were placed and 5 μL of [ 125 I] HY-3-24 (700 kBq) was added.The mixture was vigorously shaken for 1 min followed by standing for 20 min to completely separate the layers.Five 1 mL of aliquots were taken from each phase and the radioactivity of each fraction was measured by a Wizard2 Automatic Gamma Counter.LogD 7.4 was measured in quintuplicate and calculated as the logarithm of the ratio of radioactivity in organic phase to that in water phase.

Specific binding of [ 125 I]HY-3-24 on rat ventral striatum
Frozen rat brains (Sprague-Dawley rat, Male, 6-8 weeks old) were obtained from Innovative research, Inc. (Novi, Michigan, United States).The ventral striatum was isolated by dissection on ice.Cold homogenization buffer (HB, 20 mM Tris-HCl, 5 mM MgCl 2 , 1 mM EDTA, pH7.5) was added and the tissue homogenized by mortar and pestle on ice.Homogenized tissue was centrifuged at 10,000 × g, 4°C for 30 min.Supernatant was removed and pellet was mixed with cold HB buffer by vortex.This process was repeated two times to get rat ventral striatal homogenates; and protein amount was determined using a BCA protein assay kit.Homogenized tissue homogenates were stored at −80°C until use.
For saturation binding study, [ 125 I]HY-3-24 (0.001-1.6 nM concentration range) was incubated with 100 μg/100 μL of homogenized protein at RT for 60 min.Non-specific binding was defined the presence of 10 μM (+)-butaclamol, and total reaction volume was 200 μL.The reaction was finished by adding cold buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.5), and [ 125 I]HY-3-24 bound protein was filtered by 24-well harvester (Brandel Inc., Maryland, United States) through CF/C filter soaked with 1% PEI, and washed three times with 4 mL cold buffer.The collected filter was counted by gamma counter and data were analyzed by PRISM 9.The values of B max and K d were obtained by three individual experiments.

Competition binding study of [ 125 I]HY-3-24
PD128907, (+)-PHNO, quinpirole, SCH23390, raclopride, and eticlopride were prepared in concentrations ranging from 10 −5 to 10 −11 M for competition binding assay with [ 125 I]HY-3-24.Test compounds were mixed with 0.2-0.3nM of [ 125 I]HY-3-24 and rat ventral striatal membranes (100 μg/100 μL) were added.10 μM of (+)-butaclamol was used for determination of non-specific binding.The final reaction volume was 200 μL and the assay mixture was incubated at RT for 60 min.[ 125 I]HY-3-24 bound protein was filtered through CF/C filter by a Brandel 24-well harvester.The filter was washed with cold buffer (3 times) and counted by gamma counter.The value of K i was calculated using nonlinear regression analysis by PRISM 9 and the results was presented mean ± SD by three individual experiments.

In vitro autoradiography
Regional brain distribution of [ 125 I]HY-3-24 binding was determined by in vitro autoradiography using Sprague-Dawley rat and non-human primate brains.A Cryotome (Leica Biosystems, Germany) was used for sectioning frozen brains by coronal and/or sagittal direction, and the thickness of tissue was 10-20 μm.Sectioned slides were stored at −80°C until use.The adjacent brain slides were warmed to RT for 20 min, the slides were incubated with cold reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 7.5) at RT for 10 min, and the buffer was discarded.The slides were then incubated with 0.10.3 nM of [ 125 I]HY-3-24 at RT for 1 h.For non-specific binding, 2 μM of (+)-butaclamol was used.After incubation, the slides were washed with cold buffer (three times) for 2 min, then air dried at RT for 10 min.Radiolabeled brain tissues were exposed to BAS imaging plate (Fujifilm, Tokyo, Japan) for 3 days, and the result was digitized by Typhoon FLA 7000 (GE Healthcare, Illinois, United States) and analyzed by Multi Gauge V3.0.For blocking studies comparing (+)-butaclamol and raclopride, brain slides were initially pretreated with blocking agent (50 nM) for 1 h.prior to the addition of the radioligand (0.3 nM).

Chemistry
HY-3-24 and its boron-containing precursors for [ 125 I]iodine labeling were synthesized as shown in Scheme 1. 5-Iodo-2,3dimethoxybenzoic acid (1) was conjugated with a Boc-protected pendant amine to give 2; the Boc was removed using TFA to introduce the propyl linker in 63% yield for two steps (Kim et al., 2023).The linker was introduced in a good yield (76%) by N-alkylation of 3 with N-(3-bromopropyl)phthalimide.The phthalimide 4 was converted to the free amine 5 using hydrazine.After preparing 4-(pyridine-4-yl) benzoyl chloride in situ, a solution of 5 in CH 2 Cl 2 was added with a base as an acid scavenger.HY-3-24 was successfully synthesized with a satisfactory yield (73%).Two different precursors, pinacol boronic ester 7 and boronic acid 8 were synthesized to evaluate the optimal conditions for radioiodine labeling.For the insertion of boron, Miyaura borylation was performed using bromine functionalized substrate (Kim et al., 2023).Although 7 and 8 could be synthesized under the same reaction conditions, the purification procedure was different depending on the polarity of the prepared compound.7 was purified by flash chromatography using a neutral CH 2 Cl 2 and methanol mixture as a mobile phase, whereas 8 was prepared by HPLC purification using an aqueous 0.1% TFA in water and acetonitrile.It was found that the pinacol boronic ester was easily hydrolyzed on a silica material under an acidic conditions.7 and 8 were prepared with yields of 21 and 18%, respectively.

In vitro receptor binding profiles and functional activity
Binding affinities for D2-like dopamine receptor subtypes were measured and the results are shown in Table 1 (Supplementary Figure S1).For the binding assay, transfected HEK293 cells expressing hD2/D3/D4R subtype were used; [ 125 I]IABN was used as the radioligand.The K i value of HY-3-24 for D3R was 0.67 ± 0.11 nM, and the D2R K i value was 86.7 ± 11.9 nM (~129-fold selectivity for D3R).Furthermore, HY-3-24 was not active at D4R having a K i value 1,000 nM.Thus, HY-3-24 binds selectively to the D3R over the other D2-like dopamine receptor subtypes.Radioligand binding assays were also performed using HY-3-24 with [ 3 H]pentazocine for sigma-1 and [ 125 I]RHM4 for sigma-2.HY-3-24 did not bind to sigma-1 and sigma-2 receptors (Supplementary Figure S2).
A β-arrestin recruitment assay was performed to assess D3R agonist activity and D3R antagonist activity.The EC 50 of HY-3-24 was compared with dopamine for D3R agonist activity; there was no β-arrestin recruitment when the HY-3-24 was run in the agonist mode at a concentration ranging from 10 −5 to 10 −11 M (Table 1; Supplementary Figure S3A).When run in the antagonist mode, the IC 50 value of HY-3-24 for the maximum inhibition of a dopamine (EC 80 concentration) was 1.5 ± 0.58 nM (Table 1; Supplementary Figure S3B).

Radiochemistry
Cu-catalyzed [ 125 I]iodination was performed based on the previously established method in our group (Scheme 2) (Reilly et al., 2018).Since both of pinacol boronic ester and boronic acid can be used for the precursor, the labeling was performed using 7 or 8 with different reaction conditions (Table 2).Test precursors 7 and 8 were successfully labeled with [ 125 I]iodine in an excellent radiochemical yield (RCY) within a short time (Supplementary Figure S4).Particularly, the boronic acid 8 exhibited slightly higher RCY (97 or 94% at RT or 100°C, respectively) than the pinacol boronic ester 8 (89 or 88% at RT or 100°C, respectively).In the radio-HPLC, the impurity was confirmed unreacted [ 125 I]iodine at RT and by-products at 100°C according to the retention time.When the reaction time was increased at 100°C, the by-products were significantly increased.
For the in vitro validation study, the boronic acid 8 was selected for the radiosynthesis of [ 125 I]HY-3-24.The labeling conditions for the scale-up was optimized based on the test labeling results by heating at 100°C for 10 min and incubating at RT for 30 min.The amount of 8 was increased from 0.2 to 0.4 μmol.The RCY was 54.4 ± 10.1% (n = 4) that was calculated from the radioactivity of the isolated product and not decay corrected.The radiochemical purity of [ 125 I]HY-3-24 was 99% and the molar activity was 1,400 Ci/mmol based on analytical HPLC (Supplementary Figure S5).

Determination of distribution coefficient (LogD 7.4 )
LogD 7.4 of [ 125 I]HY-3-24 was measured by the shake-flask method in quintuplicate and were determined as 2.41 ± 0.01.This LogD 7.4 value demonstrated that [ 125 I]HY-3-24 is well-balanced between solubility and permeability and suitable for drug candidate.

Pharmacological characterization of D3R binding sites in the ventral striatum
To measure equilibrium property between [ 125 I]HY-3-24 and D3R, [ 125 I]HY-3-24 was incubated with 100 μg of protein at RT and the result was observed up to 2 h of incubation.Equilibrium was observed about 30 min after reaction, and it was persisted until 2 h (Supplementary Figure S6).The equilibrium conditions were used for direct binding studies.The percentage of specific binding was over 70%, and non-specific binding was less than 10% of total binding.Saturation curve were a single phase, and Scatchard plot was linear.
Both results were indicated that [ 125 I]HY-3-24 has a single binding site in rat ventral striatal membranes.The K d value for [ 125 I]HY-3-24 was 0.34 ± 0.22 nM, and B max was 38.91 ± 2.39 fmol/mg of protein (Figure 1).The K d value was calculated by Prism 9 and the data was obtained from three individual experiments.
To confirm that the binding of [ 125 I]HY-3-24 was specific for D3R, blocking studies were conducted in rat tissue homogenates using the D3-selective antagonist SB-277,011A and the D2-selective antagonist L-741,626 (Figure 3).There was complete blocking of [ 125 I]HY-3-24 by SB-277,011A at 10 and 100 nM (i.e., reduced to the same level of nonspecific binding), whereas there was no displacement of [ 125 I] HY-3-24 binding to rat striatal membranes at a concentration of 2.4 nM, the reported Ki value for displacing [ 3 H]spiperone to D2 receptors (Kulagowski et al., 1996).These data confirm that [ 125 I] HY-3-24 is highly selective for D3R vs. D2R.
To explore potential species differences, in vitro autoradiography was performed in rhesus monkey brain sections (Figure 5).The binding of [ 125 I]HY-3-24 was observed on caudate nucleus (Cd), putamen (Pu), NAc, and ICj with high density (Figure 5A).As in the rat autoradiography studies, blocking with 2 μM of (+)-butaclamol demonstrated a low level of non-specific binding of the radioligand (Figure 5B).

Discussion
The dopamine D3R is considered an important CNS target since a change in density of D3 receptors is thought to be involved in a variety of neurological and psychiatric disorders.The availability of high affinity radioligands for the D3R that have low binding to D2R and D4R and can compete with endogenous dopamine would be a useful tool for studying the change in density of D3R both in vitro and in vivo.However, efforts to find highly D3-selective radioligands that can study D3 receptor expression in vitro and in vivo has been limited by their moderate to high binding to the D2R (Burris et al., 1995;Vile et al., 1995;Luedtke et al., 2000;Xu et al., 2009) or poor competition with endogenous dopamine (Schotte et al., 1996;Mach and Luedtke, 2018;Hsieh et al., 2021).Poor competition with endogenous dopamine limits the ability of a radioligand to image D3R both in vitro  Frontiers in Neuroscience 08 frontiersin.organd in vivo.In a recent study, our group reported a structure-activity relationship (SAR) study that led to the identification of a high affinity D3-selective ligand that was potent in inhibiting the ability of dopamine binding in a β-arrestin recruitment assay (Kim et al., 2023).These data suggest that this ligand is capable of competing with dopamine for binding to the D3R both in vitro and in vivo.The goal of the current study was to develop a D3-selective radioligand for in vitro assays based on this novel scaffold.The generation of this radioligand involved a slight modification of our lead compound by replacing a Br-group with an I-group for radioiodination with I-125.
In this study, 125 I was chosen as the radionuclide because of its high specific activity relative to tritium, and its shorter half-life, which simplifies radioactive waste disposal.Therefore, [ 125 I]HY-3-24 was prepared in over 99% of radiochemical purity within 70 min, and the ability of this new radioligand for targeting the D3R was evaluated in a panel of in vitro binding studies.
The selectivity of HY-2-34 for D3R vs. D2R and D4R was initially characterized by in vitro binding assays using engineered cells and the non-selective radioligand, [ 125 I]IABN.The results revealed that HY-2-34 has sub-nM affinity for D3R, and significantly greater than 100-fold selectivity compared to D2R; the affinity for D4R was negligible (Table 1).The selectivity of [ 125 I]HY-3-24 for D3R vs. D2R was also confirmed by conducting in vitro blocking studies in rat striatal homogenates using antagonists selective for the D3R (SB-277,011A) and D2R (L741,626) (Figure 3).HY-2-34 was also found to be an antagonist by using a β-arrestin recruitment assay.The high potency of HY-2-34 for inhibiting dopamine in the antagonist mode of the assay indicates that this compound has the ability to compete with dopamine for binding to the D3R, a key factor for imaging the D3R both in vitro and in vivo.
According to previous studies, ligands targeting D3 receptors often show cross-reactivity sigma receptors (Wallace and Booze, 1995;Huang et al., 2001).Therefore, sigma receptor binding studies was performed and demonstrated that HY-2-34 has a low affinity for sigma-1 and sigma-2 receptors.Likewise, the localization of [ 125 I] HY-3-24 binding site in vitro autoradiography was indicated very low density in cortex and hippocampus noted for highly expressed regions for sigma receptors (Gundlach et al., 1986;Kim et al., 2022).
The possibility of off-target binding to other GPCRs was also a main concern for HY-2-34 because of the conformational flexibility of the molecule.Therefore, HY-2-34 was screened for binding to a panel of GPCRs such as serotonin, histamine, and opioid receptors by PDSP (Supplementary Table S1).High binding affinity was observed only at dopamine D3R, and HY-2-34 was not active or moderately active with over 100 nM affinity for the other GPCRs with the exception of dopamine D2R (K i = 43 nM) and 5-HT 2B receptors (K i = 38 nM).The affinity dopamine D3R was also investigated to be a K i value of  1.2 nM.The slight differences observed between PDSP and in-house methods for dopamine D2R and D3R are likely attributed to the radioligand used in the assay.While PDSD employed [ 3 H] N-methylspiperone, our lab utilized [125 I]IABN, which displayed negligible non-specific binding (Luedtke et al., 2000;Reilly et al., 2019).Regarding the 5-HT 2B receptor, its localization includes the dorsal hypothalamus, frontal cortex, medial amygdala, and meninges.The low binding of [ 125 I]HY-3-24 to these brain regions in the in vitro autoradiography studies suggests that off-target binding to 5-HT 2B is not likely to be a problem with this radioligand.Since dopamine D3R was known to exist with high density in rat ventral striatum (Levesque et al., 1992;Bancroft et al., 1998), the equilibrium property of [ 125 I]HY-3-24 to D3R was measured on ventral striatum homogenized by saturation experiment.Under the same conditions, Scatchard studies were conducted to measure the K d value of [ 125 I]HY-3-24, which was found to be 0.34 ± 0.22 nM.Competition studies were conducted using commercially-available D3R ligands to determine the pharmacological profile of [ 125 I]HY-3-24.Eticlopride had the highest affinity for competing with [ 125 I]HY-3-24 to D3R (K i = 0.25 nM), which is consistent with literature value for eticlopride at the D3R (Keck et al., 2015).Raclopride has a 10-fold lower affinity for displacing [ 125 I] HY-3-24 to ventral striatum followed by the D1 antagonist, SCH23390, which has a 1,000-fold lower potency in this assay.Among the tested agonists (±)-PHNO was the most potent in displacing [ 125 I]HY-3-24 (K i = 1.72 nM), followed by PD128907 (K i = 7.65 nM) and Quinpirole (K i = 7.65 nM).The rank order on the K i values of the antagonists and agonists is consistent with their affinity for the D3R (Pugsley et al., 1995;Keck et al., 2015;Doot et al., 2019).
The regional distribution and density of the dopamine D3R in brain has been of great interest for many years and was initiated by the hypothesis that D3R may be important target for neurological and neuropsychiatric disorders (Sokoloff et al., 1990).Over the past 3 decades, there have been several attempts to quantify the density of the D3R in the CNS, but these studies utilized radioligands that had a suboptimal selectivity for the D3R vs. D2R (i.e., <100-fold) (Burris et al., 1995;Vile et al., 1995;Luedtke et al., 2000;Xu et al., 2009;Zhen et al., 2010).The high selectivity of HY-2-34 for D3R vs. D2R (~129fold) suggests that a radioiodinated version of this compound may be a useful radioligand for in vitro binding studies and in vitro autoradiography studies.The low off target binding of this ligand vs. other radiolabeled D3R ligands (e.g., [ 3 H]WC-10), also make this an attractive radioligand for in vitro autoradiography studies.The present results using [ 125 I]HY-3-24 in rat brain sections confirmed that the highest density of D3R in this species was in the ICj followed by the NAc including ICjM, which is consistent with earlier observations (Schotte et al., 1996;Bancroft et al., 1998).Furthermore, our quantitative results in non-human primate brain demonstrate that the density of D3R is much higher in the striatal regions (caudate and    putamen) than the striatum of rodent brain.These results are consistent with our previous studies with [ 3 H]WC-10 (Xu et al., 2009(Xu et al., , 2010)).The advantage of [ 125 I]HY-3-24 over [ 3 H]WC-10 is that its high selectivity for the D3R avoids the uses of a duo-radioligand study with [ 3 H]raclopride and a complex calculation to tease the density of the D3R from the D2R.We are currently conducting in vitro autoradiography studies with [ 125 I]HY-3-24 in postmortem brain sections in a variety of CNS disorders.

Conclusion
In summary, our results indicate that HY-2-34 is a novel D3R selective ligand having the following features: (1) sub-nanomolar affinity (K i = 0.67 ± 0.11 nM); (2) high selectivity for D3R (>10-fold for D3R vs. D2R); (3) a high potency as a D3R antagonist (IC 50 = 1.5 ± 0.58 nM); and (4) low affinity to other GPCRs.Furthermore, [ 125 I]HY-3-24 appears to be a novel radioligand exhibiting high binding affinity and specificity at D3R: (1) K d = 0.34 ± 0.22 nM and B max = 38.91 ± 2.39 fmol/mg protein on rat ventral striatum membrane homogenates; and (2) specific binding to NAc including ICjM and ICj in rat and NHP brain tissues.Based on all results, it is anticipated that [ 125 I]HY-3-24 is promising candidate for use as the specific D3R radioligand in in vitro binding and in vitro autoradiography studies.

FIGURE 4
FIGURE 4 Specific binding site of [ 125 I]HY-3-24 in rat brain.Sectioned brain slides (coronal direction) were incubated with ~0.3 nM of [ 125 I]HY-3-24, and nonspecific binding were determined by the presence of 2 μM (+)-butaclamol.The region of interest (ROI) in rat brain was selected and quantified by multi-gauge V3.0.The results (N = 3) are presented as a mean ± SD of three different rat brain samples.(A) Total binding, (B) non-specific binding, (C) quantification of results between total binding and non-specific binding (Ordinary one-way ANOVA, ****p < 0.0001), and (D) comparison between STr, NAc including ICjM, and ICj (Ordinary one-way ANOVA, **p < 0.005).